人粒细胞中嘌呤核苷磷酸化酶的特性及其对脱氧核苷的代谢

Characterization of purine nucleoside phosphorylase from human granulocytes and its metabolism of deoxyribonucleosides.

作者信息

Wiginton D A, Coleman M S, Hutton J J

出版信息

J Biol Chem. 1980 Jul 25;255(14):6663-9.

Abstract

Purine nucleoside phosphorylase (EC 2.4.2.1) has been purified 715-fold from human granulocytes by formycin B affinity chromatography. The purified enzyme has a subunit molecular weight of 32,800 +/- 1,300 and an estimated native molecular weight of 102,000, which indicates a trimeric subunit structure. The purified enzyme migrates as a single band upon denaturing and nondenaturing polyacrylamide gel electrophoresis. Its amino acid composition has been determined. Isoelectric focusing of the purified enzyme produces three protein bands with isoelectric pH values of 5.8 to 6.1. The purified enzyme catalyzes the phosphorolysis of inosine, deoxyinosine, guanosine, and deoxyguanosine with apparent Km values of 0.21 mM, 0.32 mM, 0.21 mM, and 0.24 mM, respectively. The ribonucleosides and deoxyribonucleosides of adenine and the pyrimidines are not substrates. There are no major differences in metabolism of ribonucleosides and deoxyribonucleosides. Purine nucleoside phosphorylase activity is inhibited by formycin B in a competitive manner and by adenine in a mixed noncompetitive manner. Antiserum to the purified protein has been prepared and is useful in the immunochemical assay of phosphorylase over the range 10 to 100 ng of protein.

摘要

通过间型霉素B亲和层析法，已从人粒细胞中纯化出嘌呤核苷磷酸化酶（EC 2.4.2.1），纯化倍数达715倍。纯化后的酶亚基分子量为32,800±1,300，估计天然分子量为102,000，表明其为三聚体亚基结构。纯化后的酶在变性和非变性聚丙烯酰胺凝胶电泳中均迁移为单一蛋白条带。已测定其氨基酸组成。对纯化后的酶进行等电聚焦，产生三条蛋白条带，等电点pH值为5.8至6.1。纯化后的酶催化肌苷、脱氧肌苷、鸟苷和脱氧鸟苷的磷酸解反应，其表观Km值分别为0.21 mM、0.32 mM、0.21 mM和0.24 mM。腺嘌呤的核糖核苷和脱氧核糖核苷以及嘧啶不是该酶的底物。核糖核苷和脱氧核糖核苷的代谢没有显著差异。嘌呤核苷磷酸化酶活性受到间型霉素B的竞争性抑制和腺嘌呤的混合型非竞争性抑制。已制备针对纯化蛋白的抗血清，可用于在10至100 ng蛋白范围内对磷酸化酶进行免疫化学测定。